Mammary development in mice begins on embryonic day 10.5 (E10.5) with the formation of two mammary lines on either flank located between the fore and hind limb buds. By E11, cells in these lines coalesce into 5 pair of mammary placodes that subsequently invaginate into the underlying mesenchyme to form the mammary buds. On E16.5 the buds give rise to nascent ducts that grow down into the mammary fat pad and begin to branch. Proper embryonic mammary development relies on a series of reciprocal and sequential interactions between the epithelial cells and the surrounding mesenchymal cells. Parathyroid hormone-related protein (PTHrP) is secreted by mammary epithelial cells as soon as the placodes begin to form and it interacts with its receptor located on mesenchymal cells in order to influence the differentiation of the dense mammary mesenchyme (DMM). As a result of PTHrP signaling, the DMM maintains the mammary fate of the epithelial cells, it induces the formation of the nipple and it triggers outgrowth of the epithelial duct system. Disruption of the genes for either PTHrP or its receptor transforms the mammary mesenchyme into dermis and the mammary epithelial buds into epidermis, which, in turn, leads to a failure of mammary development at the bud stage. Conversely, overexpression of PTHrP in the developing epidermis converts the ventral dermis to mammary mesenchyme and the ventral epidermis to nipple skin. Recent data from our laboratory has demonstrated that PTHrP interacts with ventrally expressed BMP-4 in order to suppress hair follicle development and to trigger ductal outgrowth. New preliminary data demonstrate that PTHrP also regulates mesenchymal Wnt signaling within the DMM. Our working hypothesis, then, is that PTHrP acts to modulate mesenchymal cell responsiveness to Wnt and BMP signaling and that these pathways interact to mediate the various functions of PTHrP in the embryonic mammary bud. In order to investigate this hypothesis, we outline 4 Specific Aims. Aim 1 seeks to define the contribution of BMP signaling to the differentiation of the mammary mesenchyme and the initiation of ductal outgrowth. Aim 2 seeks to define the contribution of mesenchymal Wnt signaling downstream of PTHrP to embryonic mammary development. Aim 3 seeks to determine if PTHrP acts through the cAMP/PKA or PLC/PKC pathway downstream of the PTH receptor in the DMM. Aim 4 seeks to determine if PTHrP facilitates direct interactions between BMP and Wnt signaling to regulate gene expression in the DMM. Our ultimate goal is to understand better the sequential and reciprocal epithelial-mesenchymal interactions that guide embryonic mammary development and to gain insight into the mechanisms by which PTHrP modulates these interactions. PUBLIC HEALTH RELEVANCE: Breast cancer is the most common neoplasm afflicting women in the US and other developed countries. This disease causes much suffering, is responsible for a large number of premature deaths and costs the US economy a great amount each year. The source of most morbidity and mortality from breast cancer is not the primary tumor itself, which can be removed surgically, but rather metastases to the skeleton and soft tissues. Therefore understanding the mechanisms related to tissue invasion and metastases are crucial for better therapy. Normal mammary development relies on many processes such as the modulation of cell adhesion, cell proliferation, cell migration and matrix remodeling that are common to tumor invasion and the metastatic cascade. This is particularly true of embryonic mammary development, which involves the invasion of the nascent mammary epithelial ducts from one stromal compartment and into another. Our preliminary data suggest that the initiation of embryonic ductal proliferation may rely on interactions between PTHrP, Wnt and BMP signaling. All three of these growth factor pathways are also involved in breast cancer pathogenesis. Therefore, we believe that understanding how PTHrP modulates mesenchymal Wnt and BMP signaling to regulate embryonic mammary development may ultimately help us to understand the metastatic process better.